Barcode-High Resolution Melting Clearly Distinguishes Durum and Bread Wheat Varieties by Novel Primer Sets

Wheat is a vital global staple, with Triticum durum Desf. (durum wheat) primarily used for pasta and couscous, and Triticum aestivum L. (bread wheat) widely cultivated for baking. Differentiating these species, especially in processed forms, poses significant challenges due to their genetic similarity and overlapping end-use applications. This study applied Barcode-High-Resolution Melting (Bar-HRM) analysis to distinguish common durum (Cesare, Ovidio, Tiziana, Zivago) and bread wheat (Adana 99, Ceyhan 99, Dinç, Nizar) varieties in Türkiye. DNA was extracted separately from seeds, flours, and fresh leaves to ensure successful recovery, with seed-derived DNA used for HRM analysis. The Internal Transcribed Spacer (ITS) region was amplified via PCR, revealing two polymorphic sites in ITS1 and 20 in ITS2, with 14 sites differentiating the two species. HRM primer sets targeting these regions achieved genotyping confidence levels exceeding 95%. Sequencing validated the HRM results, and phylogenetic analysis confirmed clear separation between durum and bread wheat. This rapid, reliable, and contamination-resistant method enables accurate wheat discrimination from small samples within three hours, offering significant potential for broader applications in agricultural diagnostics and food authenticity testing